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Transcript
Viral Detection
By: Douglas Tran
Endogenous Viral Complexes with long RNA
cosediment with the agent of Ceutzfeldt-Jacob
Disease
 Past research
 States Prion hypothesis and past research of no past
viral genetic material detected
 States the fact that mitochondrial DNA has been found
in subsequent studies
 States that long RNAs cores were also detected
 Very similar to retroviral cores
Endogenous Viral Complexes with long RNA
cosediment with the agent of Ceutzfeldt-Jacob
Disease
 CJD Retroviral Theory
 Strain variation, exponential replication, tissue
specificity, latency, resistance to treatment, and noninflammatory response
 LTRs detection experience found LTRs present in
infectious samples
 Endogenous retroviral intracisternal A particle (IAP)
genome were identified by using cDNAs that were
created from the LTRs of infectious factions
Endogenous Viral Complexes with long RNA
cosediment with the agent of Ceutzfeldt-Jacob
Disease
 IAP RNA cosediement found (The RNA that is being
detected)
 IAP are a class of retrovirus
 In Infectious sample
 In non-infectious sample
 IAP are highly resistant to forms of treatment like SDS and
chaotropic salts
 High resistance is due to IAP gag proteins
 Isolation of this gag protein would help solidify the idea of
virus particle, not just random IAP RNA
Endogenous Viral Complexes with long RNA
cosediment with the agent of Ceutzfeldt-Jacob
Disease
 Source of materials and purification of CJD infectivity
 Syrian hamster IAP genome
 Polyclonal antibodies that bind the IAP gag protein
 12 CJD infected hamster brains
 Purification: samples made devoid of PrP-res
 Samples also treated with nuclease (allows for isolation of
nuclease resistant genetic material)
 Extraction of RNA
 Elimination of DNA in sample via various methods (DNAase)
Endogenous Viral Complexes with long RNA
cosediment with the agent of Ceutzfeldt-Jacob
Disease
 RNA/PCR amplification of IAP sequences
 Usage of various IAP primers for the production of
subsequent cDNA syntheses
 IAP RNA from cells -> cDNA-> DNA amplification via IAP
primer
 Probe hybridization to Southern Blots
 DNA probes
 Generated from recombinant clone of Syrian Hamster IAP
 Western blot
 Polyclonal rabbit anti-mouse IAP gag antibodies
Endogenous Viral Complexes with long RNA
cosediment with the agent of Ceutzfeldt-Jacob
Disease
Endogenous Viral Complexes with long RNA
cosediment with the agent of Ceutzfeldt-Jacob
Disease
Endogenous Viral Complexes with long RNA
cosediment with the agent of Ceutzfeldt-Jacob
Disease
• Discussion:
• Was able to isolate both IAP RNA and RNA gag in
infectious samples
• Both are resistance to degradation of
nuclease, which shows characteristic of
retroviral
• Results refuted the assumption of prion
infection being devoid of nucleic acid
• ~6kb IAP sequences
• No clear evidence of a CJD specific sequence of
IAP RNA
• If IAPs are involved with the CJD nucleic
acid, it is either co-packed in the core or uses
IAP products to proliferate
• Second theory: Completely independent CJD viral
complex, only similar to IAP
• Supported by presence of gag-like proteins
Viral Particles are required for infection in
neurodegenerative Creuzfeldt-Jakob disease
 States the assumption of virus infectivity theory
 Characteristic of the CJD infectious agent
 Core-like viral density of 1.27-1.28 g/cc
 Viral size of 120S and a diameter of ~30nm
 >99% of starting prion protein can be separated from the viral agent
 Primary investigation: Separation and isolation of the primary
components of the viral protein (testing different methods)
 PrP
 Nucleic acid-binding proteins
 Gag protein
Viral Particles are required for infection in
neurodegenerative Creuzfeldt-Jakob disease
 Method
 Centrifuge isolation of supernatant and pellets after
elimination of the majority of PrP
 Immunoblots (Western blot method used for protein
isolation)
 Polyclonal antibodies
Viral Particles are required for infection in
neurodegenerative Creuzfeldt-Jakob disease
Viral Particles are required for infection in
neurodegenerative Creuzfeldt-Jakob disease
Viral Particles are required for infection in
neurodegenerative Creuzfeldt-Jakob disease
Viral Particles are required for infection in
neurodegenerative Creuzfeldt-Jakob disease
Viral Particles are required for infection in
neurodegenerative Creuzfeldt-Jakob disease
 Results show that a specific nucleic acid and one or more
binding proteins are intrinsic components of the CJD virus
 Independently have low infectivity, but together form a
complex that is the main infective agent
 Infectivity is not Prp dependent,
 SDS treatment eliminates the majority of Prp, but infectivity is
still high
 Gdn-HCI treatment who greatly lower infectivity due to
elimination of the viral components
 There is still retention of PrP in Gdn-HCI samples
Nuclease resistant circular DNAs copurify with
infectivity in scrapie and CJD
 Begins paper by stating examples of virus that have
similar and/or the same characteristics of CJD viral
infection
 States theory involving PrP as the viral receptor and
that after interaction with virus it causes the
formation of PrP-res
 Lists the common flaws in Prion Hypothesis
Nuclease resistant circular DNAs copurify with
infectivity in scrapie and CJD
 Primary goal:
 Isolation and identification of circular ssDNA via
generalized detection method
 Ф29 polymerase to enhance the replication of the circular
ssDNA
 Circular DNA hypothesis is based off CJD’s similarity with
Torgue tenovirus, which has circular DNA
 It is also based on Circular DNA’s ability to cause physical
generation like the formation of mouse tumors
Nuclease resistant circular DNAs copurify with
infectivity in scrapie and CJD
 Infectious material:
 Three source that underwent PrP depletion procedures
 Murine N2a neuroblastoma (22L)
 Hamster brain (263K)
 Japanese FU-CJD patient (FU-CJD)
 Nucleic acid extractions
 Usage of Proteinase K for digestion of endogenous or added
nucleases
 Used purification method for digested genetic material
Nuclease resistant circular DNAs copurify with
infectivity in scrapie and CJD
 Ф29 polymerase and ssDNA chromatography
 Allow for the amplification of circular ssDNA from the
digested nucleic acid
 RNA and RNAase inhibited the effects of Ф29
polymerase
 ssDNA was isolated from these factors and dsDNA before
replication
 Restriction enzyme analysis, PCR and sequencing
 Digestion of the Ф29 polymerase product -> PCR
proliferation -> sequenced
Nuclease resistant circular DNAs copurify with
infectivity in scrapie and CJD
Nuclease resistant circular DNAs copurify with
infectivity in scrapie and CJD
Nuclease resistant circular DNAs copurify with
infectivity in scrapie and CJD
Nuclease resistant circular DNAs copurify with
infectivity in scrapie and CJD
Nuclease resistant circular DNAs copurify with
infectivity in scrapie and CJD
Nuclease resistant circular DNAs copurify with
infectivity in scrapie and CJD
 Demonstration of two new circular DNAs that is in
high concentration in TSE particles
 All three strains tested strongly positive for Sphinx 1.8kb
and 2.4kb, which show a clear viral plasmid structure
 Both are passed down to daughter cells
 Both are also present in uninfected brains, but the
concentration in infected brain is x2,500 higher
 Positive control: Mitochondrial DNA was used
 In essence, Sphinx DNA is viral, their actual role is up
for further investigation